1. Field Of The Invention
Protein overproducing bacterial cells are separated from less dense bacterial cells which do not overproduce protein.
2. State Of The Art
It is known to employ gene splicing technology to overproduce proteins in microorganisms into which genes are spliced that code for said proteins. For the optimal synthesis of proteins encoded by the spliced genes, it is necessary to join several deoxyribonucleic acid (DNA) pieces in a very precise manner. Since the construction of spliced DNA molecules requires multiple steps of enzymatic reactions, the chance of successful construction may be quite low. As a result, screening a large number of bacterial clones is often necessary to isolate those having the desired recombinant DNA structures. The process of this invention allows rapid segregation of bacteria having the desired construction from those which do not.
Protein production in gram-negative, enterobacteria such as E. coli using recombinant DNA technology has been demonstrated by a number of investigators. For instance, Goeddel et al., Proc. Natl. Acad. Sci. 76, pages 106 to 110 (1979), describe the use of E. coli cells carrying recombinant plasmid plBl in the synthesis of a hybrid insulin A chain-.beta.-galactosidase protein in up to 20% of the total cell protein. Cheng et al., Gene 14, pages 121 to 130 (1981), describe the bacterial synthesis of .beta.-galactosidase in up to 15% of the total E. coli protein. In both cases, the proteins synthesized form intracellular protein aggregates.
U.S. Pat. No. 3,072,538, discloses a method of separating strains of living soil bacteria capable of synthesizing poly (.beta.-hydroxybutyric acid) from other strains by density separation in an aqueous suspension. The bacteria capable of producing the polyester sink into the suspension. U.S. Pat. No. 4,138,291, discloses a method of obtaining selected bacterial strains capable of converting a carbon source into poly(D-3-hydroxybutyric acid). The selection process depends on specific weight differences. The following publications disclose the use of density gradient separation of temperature-sensitive mutants of the yeast Saccharomyces cerevisiae: Novick et al., Cell., 21, page 205 to 215 (1980); and Nishizawa et al., Abstract of the 1981 Meeting of the Molecular Biology of Yeast, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Nowhere in the literature, however, has any report been found concerning the unusual physiological property of a density increase in protein overproducing cells versus like cells which do not overproduce proteins. The discovery of this characteristic density difference allows rapid detection, identification, isolation, enrichment and recovery of protein overproducing bacteria from admixture with bacterial clones that do not have such property.